HIV-1 gp120 Conformational Transitions in Activation and Antagonism

描述(由申请人提供)：该项目解决了设计HIV-1进入抑制剂的一个主要障碍，该障碍源于病毒包膜糖蛋白gp120的高构象可变性。不同的实验观察表明，HIV-1包膜蛋白在与宿主细胞受体结合后经历了大量的构象结构。这些构象变化导致细胞受体的Env结合部位逐渐成熟，是病毒-细胞融合、细胞进入和随后的感染所必需的。此外，已鉴定出几种类型的Env抑制因子前导，它们似乎通过结合HIV-1 gp120的未连接状态并从构象上将Env蛋白包裹到抑制细胞进入的受体结合位点活性的状态来发挥作用。这一系列的观察认为，了解受体和抑制剂诱导的未连接gp120构象转变的位置和动力学的差异，可以帮助识别最有效地抑制前者而鼓励后者的抑制剂。然而，去连接状态本身，本质上是病毒包膜拮抗剂的主要结合靶点，以及从这种结构开始发生的构象变化的动力学，人们对此知之甚少。这些缺点将在目前的R21项目中得到解决，方法是使用创新的策略来跟踪从非连接形式到连接形式的Env gp120特定位点的构象变化。R21项目将有两个具体目标。在目标1中，我们将生产荧光标记的gp120变异体，其中Env蛋白被标记在不同的结构位置，预计当与已知的包膜配体结合时，构象会改变，包括CD_4以及CD_4模拟和变构抑制剂。在目标2中，我们将测量配体与单一位点标记的gp120变异体结合时，时间分辨和稳态荧光各向异性的位置特异性变化。来自不同标记位点的实验各向异性曲线将被用来定义gp120在未连接、激活和抑制状态下的构象特征。R21项目最直接的成果将是：(1)获得功能完整的gp120变异体，带有位置特定的荧光团报告标签；(2)证明通过荧光跟踪可以在gp120中空间定义的位置检测到配体诱导的构象变化。R21的后续长期研究将绘制gp120在受体激活和拮抗剂(竞争性和变构)抑制时发生构象变化的特定位置，并基于荧光筛选导致特定局部构象变化的抑制剂，从而改善对HIV-1 Env蛋白的抑制。 公共卫生相关性：该项目将建立一种创新的多学科方法，结合蛋白质工程和荧光光谱学，以扩大对HIV-1病毒外壳蛋白结构转变的理解，并长期指导识别作为抗病毒药物的进入抑制剂。

DESCRIPTION (provided by applicant): This project addresses a major barrier in designing HIV-1 entry inhibitors that stems from the high conformational variability of the viral envelope glycoprotein gp120. Diverse experimental observations have shown that the HIV-1 envelope protein undergoes large conformational structuring upon binding to its host cell receptors. These conformational changes lead to progressive maturation of Env binding sites for the cell receptors and are required for virus-cell fusion, cell entry and consequent infection. In addition, several types of Env inhibitor leads have been identified that appear to function by binding the unliganded state of HIV-1 gp120 and conformationally entrapping Env protein into states that have suppressed receptor binding site activity for cell entry. This body of observations argues that knowledge of differences in the locations and dynamics of conformational transitions of unliganded gp120 induced by receptors vs inhibitors could help to identify inhibitors that most effectively frustrate the former while encouraging the latter. Yet, the unliganded state itself, which is in essence the primary binding target for antagonists of viral envelope, is poorly understood, as are the dynamics of conformational changes that occur starting from this structure. These shortcomings will be addressed in the current R21 project by using innovative strategies to track site-specific conformational changes leading from unliganded to liganded forms of Env gp120. The R21 project will have two specific aims. In Aim 1, we will produce fluorophore-tagged gp120 variants in which the Env protein is labeled in different structural locations predicted to change conformations upon binding to known envelope ligands, including CD4 and both CD4-mimicking and allosteric inhibitors. In Aim 2, we will measure location- specific changes in both time-resolved and steady state fluorescence anisotropy upon ligand binding to single- site labeled gp120 variants. Experimental anisotropy curves derived from distinctly labeled sites will be used to define conformational signatures of gp120 in unliganded, activated and inhibited states. The most immediate outcomes of the R21 project will be (1) derivation of functionally intact gp120 variants with location-specific fluorophore reporter tags and (2) demonstration that ligand-induced conformational changes can be detected at spatially defined sites in gp120 through fluorescence tracking. Longer term research following up from the R21 will map specific sites of conformational changes in gp120 that occur upon receptor activation vs antagonist (competitive as well as allosteric) inhibition and derive fluorescence-based screens for inhibitors that induce specifically localized conformational changes leading to improved inhibition of HIV-1 Env protein. PUBLIC HEALTH RELEVANCE: This project will establish an innovative multidisciplinary approach, combining protein engineering and fluorescence spectroscopy, to expand understanding of the structural transitions of HIV-1 virus coat protein and long term guide identification of entry inhibitors as antiviral agents.